Cortisol is a steroid hormone produced by the adrenal gland in order to regulate multiple body functions ranging from glucose metabolism to immune function and inflammatory response (Petkus et al., 2006). Cortisol helps to maintain blood pressure, immune function, and the body's anti-inflammatory processes. Aggressive behavior of teenagers can be identified by detecting the cortisol in their saliva sample (Oosterlaan, J. et al., 2005). The amount of cortisol released by the adrenal glands is regulated by the pituitary gland inside the brain. When cortisol levels are low, the pituitary gland secretes the stimulating hormone adrenocorticotropin (ACTH) to prompt the adrenal glands to make more cortisol. High cortisol levels drop when the pituitary gland slows its output of ACTH (Jezova D., 2005).
Cortisone is derived from the peripheral metabolism of cortisol and lacks biological activity. The rapid interconversion between cortisol and cortisone and the altered equilibrium between these steroids may regulate glucocorticoid activity in various tissues. Children with hypoadrenalism exhibited a greater decrease in cortisol as compared with cortisone. Children with adrenal cancer exhibited normal or high values of cortisol, whereas cortisone levels were decreased and the cortisone/cortisol ratio was decreased to nearly zero (Nomura, S. et al., 1996). The simultaneous evaluation of cortisol, cortisone, and the cortisone/cortisol ratio provides a clinical clue of adrenal diseases. Glucocorticoids play an important role in determining body fat distribution, but circulating cortisol concentrations are reported to be normal in obese patients. Conversion of inactive cortisone to active cortisol through the expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) in cultured omental adipose stromal cells; the autocrine production of F may be a crucial factor in the pathogenesis of central obesity (Stewart, P. M. et al., 1999). 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) plays a crucial role in converting hormonally active cortisol to inactive cortisone, thereby conferring specificity on the mineralocorticoid receptor (Marcus Quinkler et al. 2003). The normal ranges observed for cortisol varies from laboratory to laboratory but are usually within the following ranges for blood: adults (8 A.M.): 6-28 mg/dL; adults (4 P.M.): 2-12 mg/dL; child one to six years (8 A.M.): 3-21 mg/dL; child one to six years (4 P.M.): 3-10 mg/dL; newborn: 1/24 mg/dL.
Cortisol detection is performed on patients who may have malfunctioning adrenal glands. Blood and urine cortisol, together with the determination of adrenocorticotropic hormone (ACTH), are the three most important tests in the investigation of Cushing's syndrome (caused by an overproduction of cortisol) and Addison's disease (caused by the underproduction of cortisol). This test is a measure of serum cortisol (also known as hydrocortisone), or urine cortisol, (also known as urinary free cortisol), an important hormone produced by a pair of endocrine glands called the adrenal glands.
Some disorders can be treated with synthesized cortisol, called corticosteroids. One of the main side effects of long term treatment is osteoporosis (thinning of the bones). Levels of cortisol in the body change throughout the day, never being the same. They are highest in the morning, dropping rapidly until mid-day, and gradually declining throughout the rest of the day, being lowest at night (Kurina et al., 2004). Abnormal levels of cortisol may influence certain pathological conditions such as Type 2 Diabetes, constant stress, obesity, metabolic syndrome, etc. It has even been shown that elevated levels of cortisol may be influencing the severity of epileptic seizures since the increase in the adrenocorticotropic hormone as well as cortisol levels has been documented in epileptic postictal period (Galimberti et al., 2005). Since the 1960s, glucocorticoids are used by athletes to improve their performances. Their use is restricted in sports. One of the commonly used tests to determine the cortisol levels is the saliva test (Petkus et al., 2006).
Some of the more common side effects of cortisol-like drugs includes fluid retention (oedema), thin skin, susceptibility to bruising, high or increased blood pressure, osteoporosis (thinning of the bones), bone fractures, particularly in the spine and ribs.
Symptoms of cortisol insufficiency can include: fatigue, nausea and vomiting, low blood pressure, particularly when standing up from a sitting or lying position (orthostatic hypotension), low blood sugar, shock, and coma.
Thus far, the existing methods to detect cortisol, such as the fluorimetric assay (Appel, D. et al. 2005) and reverse phase chromatography (Gatti, R., et al., 2005), are limited with respect to their sensitivity, time of analysis, and cost. Enzyme fragment complementation (EFC) technology provides a sensitive and homogeneous method for measuring analytes by enzymatically amplifying the signal. These fragments are inactive separately but, in solution, they rapidly recombine to form an active enzyme by the process of complementation.
In monitoring medical conditions and the response of patients to efforts to treat medical conditions, it is desirable to use analytical methods that are fast, accurate, and convenient for the patient. Electrochemical methods have been useful for quantifying certain analytes in body fluids, particularly in blood samples. Typically, these analytes undergo oxidation-reduction reactions when in contact with specific enzymes, and the electric current generated by these reactions can be correlated with the concentration of the analyte of interest. Miniaturized versions of analytical electrochemical cells have been developed that allow patients to monitor levels of particular analytes on their own, without the need for a healthcare provider or clinical technician. Typical patient-operated electrochemical sensors utilize a single measuring unit containing the necessary circuitry and output systems. In use, this unit is connected to a disposable analysis strip containing the electrodes and the necessary reagents to measure the electrochemical properties of a sample that is applied to the strip. The most common of these miniature electrochemical systems are the glucose sensors that provide measurements of blood glucose levels. Ideally, a miniature sensor for glucose should provide accurate readings of blood glucose levels by analyzing a single drop of blood, typically from 1-15 microliters (μL).
In a typical analytical electrochemical cell, regardless of the size of the system, the oxidation or reduction half-cell reaction involving the analyte either produces or consumes electrons. This electron flow can be measured, provided the electrons can interact with a working electrode that is in contact with the sample to be analyzed. The electrical circuit is completed through a counter electrode that is also in contact with the sample. A chemical reaction also occurs at the counter electrode, and this reaction is of the opposite type (oxidation or reduction) relative to the type of reaction at the working electrode. See, for example, Fundamentals of Analytical Chemistry, 4th Edition, D. A. Skoog and D. M. West; Philadelphia: Saunders College Publishing (1982), pp 304-341.
The basic components of an electrochemical sensor are a working electrode (also referred to as a sensing electrode, measuring electrode, or anode), a counter electrode (also referred to as a cathode), and usually a reference electrode. These electrodes can be enclosed in a sensor housing in contact with an electrolyte (normally an aqueous solution of strong inorganic acids such as sulfuric or phosphoric acid). When a target molecule is detected, the cell generates a small current proportional to the concentration of the target molecule in the sample. In its simplest form, an electrochemical sensor includes a diffusion barrier, a working electrode, a counter electrode, and an electrolyte. In an environment free of chemically reactive molecules, oxygen diffuses into the cell and adsorbs on both electrodes. The result is a stable potential between the two in which little and, theoretically, no current flows. When a chemically reactive molecule passes through the diffusion barrier it is either oxidized (accepts oxygen and/or gives up electrons) or reduced (gives up oxygen and/or accepts electrons), depending upon the target molecule. The resulting potential difference between the two electrodes causes a current to flow.
The working electrode is typically on the inner face of a diffusion barrier such as a membrane (e.g., Teflon® membrane) that is porous to the target molecule, but impermeable to the electrolyte. The target molecule diffuses into the sensor and through the membrane to the working electrode. When the target molecule reaches the working electrode, an electrochemical reaction occurs; either an oxidation or reduction depending on the type of sample or target molecule.
For example, carbon monoxide may be oxidized to carbon dioxide, or oxygen may be reduced to water. An oxidation reaction results in the flow of electrons from the working electrode to the counter electrode through the external circuit; and conversely a reduction reaction results in flow of electrons from the counter electrode to the working electrode. This flow of electrons constitutes an electric current, which is proportional to the target molecule concentration. When carbon monoxide, a reducing gas, diffuses to the sensing electrode, it is oxidized causing the potential of the sensing electrode to shift in a negative (cathodic) direction. The more modern form of cell utilizes a reference electrode. This electrode has a stable potential from which no current is drawn. It is used to eliminate interference from side reactions with the counter-electrode. In addition, it allows the sensing-electrode potential to be biased with respect to its rest potential. Biasing is one method of controlling sensitivity to a particular target molecule. In order to provide for extended storage, a shorting clip can be connected across the working and reference terminals. This short maintains the electrodes at the same potential and keeps current from flowing through the cell. The electrochemical cell typically includes a casing containing an electrolyte liquid or gel and three electrodes. The top of the casing has a membrane permeable to the target molecule as well as a gas capillary. The electrodes are carefully constructed to provide maximum sensitivity and long life, through a electrode construction which allows more surface area. This allows a larger signal, a quicker response and permits a smaller volume of electrolyte to provide the same life available from large sensors. Each cell is constructed using special filters, electrodes, and electrolytes to make the cell as specific as possible. Electronics should provide appropriate bias current to eliminate interfering sensitivity. Because electrochemical reactions, like all chemical processes, are temperature dependent, electrochemical sensors often incorporate a sensitive temperature sensor which the electronics use to compensate for temperature variations.
The electronics in the instrument detects and amplifies the current and scales the output according to the calibration. The instrument usually displays the target molecule concentration in, for example, parts per million (PPM) for toxic gas sensors and percent volume for oxygen sensors.
There is a need for miniaturized electrochemical systems with improved sensitivity to the concentration of target molecules in samples. It is desirable for miniaturized electrochemical strips to contain independently optimized electrodes having high conductivities.